Postangioplasty restenosis is a localized hyperproliferative disorder that develops following a non-surgical procedure related to atherosclerotic plaque. Thus, the treatment of an atherosclerotic lesion by angioplasty very frequently (up to 50% of the cases in some studies) results in restenosis as a result of mechanical injury to the arterial wall.
The proliferation of smooth muscle cells (SMC) in the vascular wall is a key event in the development of atherosclerosis and in restenosis after angioplasty, as well as in the failure of coronary by-pass procedures. SMCs are normally quiescent in the vascular wall, but as a result of a lesion that destroys vascular endothelium, they come into contact with blood growth factors. Unlike cardiac muscle cells and skeletal muscle cells, in response to different growth factors, SMCs can become dedifferentiated and their proliferative cycle can be set in motion again. These phenotypical changes in SMCs are the result of profound changes in the expression of numerous genes. As an example, the expression of early genes, which are involved in the proliferation of cells, such as c-fos or c-myc, is remarkably increased, although the expression of other genes with a structure such as specific smooth muscle alpha-actin, as well as certain isoforms of myosin undergo negative regulation.
Although it has been clearly established that terminal differentiation of skeletal muscle cells is regulated by so-called myogenic transcription factors, such as MyoD, very few of the factors involved in the reversible differentiation of SMCs are known as yet. Recent studies have revealed the existence of transcription factors that have a homeobox in different tissues of the cardiovascular system. Thanks to their homeobox, these factors recognize specific DNA sequences in the promoter regions of their target genes, and thus, they intervene in the regulation of cellular differentiation, proliferation or migration. One of these factors, gax (Growth-Arrest-Specific Homeobox), is expressed in different cardiovascular tissues, including the SMCs of the vascular wall. The gax gene was initially identified from rat aorta obtained from a cDNA bank. It encodes for a protein with 303 amino acids. Its sequence was characterized and its cDNA was cloned (Gorski et al., Mol. Cell. Biol. 1993, 6, 3722-3733). The human gax gene has also been cloned and sequenced (David F. Le Page et al., Genomics 1994, 24, 535-540). It encodes for a protein with 302 amino acids, which is shown below (SEQ ID NO. 16). The gax gene has certain properties that are similar to gas and Gadd genes since it seems to control the G0/G1 transition of the cell cycle. Thus, levels of gax mRNA are reduced in rat VSMC [vascular smooth muscle cells] by a factor of 10 after two hours of exposure to PDGF [platelet-derived growth factor] (Gorski et al., Mol. Cell. Biol. 1993, 6, 3722-3733). The expression of the gax gene is thus repressed during the mitogenic response of VSMC. Another characteristic of the gax gene is based on its specificity of expression. In fact, in the adult rat, the gax gene is expressed primarily in the cardiovascular system (the aorta and the heart). Northern Blotting did not determine the presence of gax mRNA in the liver, the brain, the stomach, and skeletal muscle. Moreover, the gax gene belongs to the family of homeotic genes. These genes encode for transcription factors which include consensus sequences (or homeodomains) that recognize specific regions of DNA (or homeoboxes) (review: Ghering et al., Cell, 78: 211-223, 1994). The homeodomain of rat gax protein is between amino acids 185 and 245. Interestingly, the homeotic genes identified to date are involved in the control of cell differentiation/growth during embryogenesis, which strengthens the therapeutic potential of the gax gene (review: Lawrence et Morata Cell 78: 181-189, 1994: Krumlauf, Cell 78: 191-201, 1994).
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One characteristic of GAX is that it undergoes a negative regulation as soon as SMCs proliferate, whether in vitro in response to growth factors, or in vivo following a lesion of the vascular wall endothelium. This repression is reversible because when the SMCs are made quiescent by deprivation in serum, in vitro, GAX expression resumes. Recent studies conducted in our laboratory showed that high-level expression of GAX in SMCs by an adenoviral vector blocks their proliferation FR 95/04234 (ST95022).
Postangioplastic restenosis following mechanical injury represents the most frequent failure factor (50% of the cases in some studies). Since SMC proliferation is a key element in this phenomenon, and since it is known that it has a regulatory role in GAX proliferation, the latter seems to be a therapeutic gene of choice for the preventive treatment of the vascular wall after angioplasty FR 95/04234(ST95022).
However, the GAX mechanism of action is still not very well documented. The target DNA sequences of the GAX homeobox still have to be identified. Moreover, as yet there have been no studies of the function of the different GAX domains. Finally, the identification of the functional partners of GAX is an important facet which will permit us to define the molecular cascade on which GAX depends or of which it is the source.